This invention relates, inter alia, to a method of altering one or more characteristics of mammalian endometrial tissue.
Endometrial Physiology
Two major events are required for the embryo to become established in the mammalian uterus: firstly, the preparation of the endometrium so that it is receptive to the presence of a blastocyst which can then implant and acquire nutritive support through the formation of the placenta; and secondly, the modification of myometrial activity which must become quiescent and thereby allow the blastocyst to become resident within the uterine cavity without the danger of expulsion. Both these events are controlled by the action of the hormones of pregnancy, of which oestrogens and progesterone are particularly important. These steroid hormones act on the endometrium and myometrium through their receptors which are located in the nucleus of target cells. Once activated, the steroid-nuclear receptor complex interacts with specific regions within the DNA to stimulate, repress or de-repress genes that code for proteins and polypeptides such as enzymes or growth factors.
The initiation of implantation is brought about by a cascade of biochemical and biophysical changes. Adhesion molecules (e.g. CAM 105) have been implicated in the early stages of attachment of the blastocyst to the wall of the uterus. Afterwards, the blastocyst and endometrium adopt various stratagems to improve intimacy between fetal and maternal tissues. In ungulates, trophoblast cells which form the outermost layer of the blastocyst migrate into the uterine epithelium with which they subsequently fuse. Cell migration is carried a step further in women because it is not only isolated or specific cell types that migrate but large areas of trophoblast which insinuate between the uterine epithelial cells. In order to do this, some of the trophoblast cells fuse together to form a syncytium. The process is very rapid and the embryo becomes established in the uterine tissues without much apparent degeneration in the uterine epithelium. In some species the process of implantation is delayed, either to await environmental cues which ensure the young are born at favourable times of the year, or by physiological factors such as lactation so that the mother has finished weaning the previous litter before the next pregnancy becomes fully established.
The preparation of the uterus for implantation is regulated by the secretion of ovarian hormones. The transport of the fertilised egg through the oviduct has to be precisely timed so that it arrives in the uterus at the correct time of development and when the uterus is in a fit condition to receive it. Under most conditions the uterus is hostile to the embryo, more hostile in fact than some other areas of the body. The epithelial lining of the uterus is, under most conditions, resistant to attachment and invasion by trophoblast and it is only under very precise hormonal states that this resistance is relaxed.
In mice and rats unmated animals do not have a full oestrous cycle because they do not form a normal secretory corpus luteum which produces increasing amounts of progesterone. If mating occurs at oestrus, a time when high levels of oestrogens are secreted by the ovarian follicle from which the ovum is shed, a corpus luteum will form in the place of the ruptured follicle, rising concentrations of progesterone are then secreted, implantation occurs and pregnancy progresses (length, 21 days). If an infertile mating occurs, similar events occur except that the corpus luteum only lasts about 11 days and pseudopregnancy is curtailed.
The cellular and biochemical changes that take place in the endometrium have been most thoroughly studied in the mouse and the rat, though information about these aspects in women has increased substantially in recent years. The endometrium in all species is made up of three main tissuesxe2x80x94luminal epithelium, glandular epithelium and stroma. Cell proliferation occurs at different times in the three tissues. Luminal cells proliferate just before oestrus (proestrus) under the influence of the rising levels of oestrogens produced by the follicles in the ovary. By day 1 of pregnancy (day of copulation plug in rodents) they have ceased division but then undergo a second, though smaller, burst of activity on day 3. Glandular cells show most activity on day 4 and then decline. Stromal cells do not proliferate until day 4 but thereafter, under the influence of progesterone, they reach high levels of proliferation by day 5. In women, less is known of these changes which presage the process of implantation but there appears to be peak proliferation in epithelial cells during the follicular phase of the cycle and in stromal cells during the luteal or secretory phase, as in the mouse and rat.
The purpose of endometrial cellular proliferation is not fully resolved. It is believed to prepare the endometrium for implantation by increasing the number of cells that will serve a nutritive and secretory function (glandular epithelium) and that participate in the very early stages of placentation (decidualization). As a prerequisite of successful implantation, cell mitosis may progress towards cellular differentiation and therefore plays a crucial role in the early events of the establishment of pregnancy. Evidence in support of this role is the endometrial production of growth factors (mitogens), cytokines and nuclear oncogenes. Many of these compounds are produced in increased concentrations in response to ovarian hormones acting through their receptors.
Amongst growth factors, much attention is currently given to epidermal growth factor (EGF), heparin binding epidermal growth factor (HBEGF), amphiregulin and insulin-binding growth factors (IGF-I and IGF-II). Evidence for the importance of the local (paracrine) action of at least one of these growth factors, amphiregulin, has been provided by recent experiments in mice. Inhibition of the implantation-specific and progesterone-regulated gene for amphiregulin was achieved by the anti-progestin, RU486, and this resulted in the prevention of implantation (Das et al. Molecular Endocrinology 9, 691-705, 1995).
Amongst the cytokines, leukaemia inhibitory factor (LIF) and colony-stimulating factor (CSF), which are also produced by the mouse uterus at the time of implantation, have been found from gene knockout studies to be indispensable, demonstrating that their removal is incompatible with implantation and normal placentation (Stewart et al. Nature 359, 76-79, 1992: Pollard et al. Developmental Biology 148, 273-283, 1991).
Amongst the nuclear oncogenes, levels of c-jun and c-fos (which are early indicators of gene transcription) increase in the uterus after oestrogen administration, and are inhibited by progesterone.
Important differences exist between various species in the extent of trophoblast invasion at the time of implantation. In women, the early trophoblast is highly invasive whereas in pigs, which have a non-invasive form of implantation, the endometrial epithelium is never breached throughout the three month gestation period. Failure of implantation in both these species is high, reaching about 60 and 30%, respectively. The reasons for this high rate of failure are complex and incompletely understood. In women, about half the loss is attributable to genetic abnormalities but in pigs, as in other ungulates where the loss is also high, genetic defects only account for a few percent of the total.
After implantation failure in women a fall in progesterone secretion causes bleeding, as at the end of the normal menstrual cycle; this does not occur in most other animals. Disorders of menstruation, as well as of implantation, are common. In addition menstrual bleeding, either as a consequence of sequential hormonal therapy, or in conjunction with continuous combined hormone replacement therapy or progestin-only long-acting contraceptives, is a significant cause of ill-health in women. The underlying reasons for this bleeding are the focus of many current studies into biochemical (e.g. prostaglandins, enzymes, polypeptides and proteins, vasoactive compounds such as platelet-activating factor PAF, and vascular endothelial growth factor VEGF) and cellular mechanisms (e.g. migrating cells homing to the uterus that produce immunosuppressive compounds).
Current understanding of reproductive processes largely centres on the control of steroid hormone production and the actions of these hormones on their target tissues. However paracrine and autocrine factors are increasingly seen to be key mediators of reproductive function, albeit interacting with steroids (Benton, 1991 Current Opinion in Cell Biology 3, 171-175: Rozengurt, 1992 Current Opinion in Cell Biology 4, 161-165; Tartakovsky et al., 1991 Developmental Biology 146, 345-352; Robertson et al., 1992 Current opinion in Immunology 4. 585-590; Smith, 1994 Human Reproduction 9, 936-946; and Tabibzadeh, 1994 Human Reproduction 9, 947-967). The clearest example of this is seen in the ovariectomized mouse. In this model the uterus undergoes marked growth in response to a single dose of estradiol. in this effect can be blocked by anti TGFxcex1 antibody (TGF is xe2x80x9ctransforming growth factorxe2x80x9d) suggesting that the mitogenic effects of estrogen in this tissue are mediated by TGFxcex1 (Nelson et al., 1992 Endocrinology 131, 1657-1664).
Consequently medical intervention in Gynaecology is largely based on steroidal/antisteroidal regulation of the uterus (Yen and Jaffe, 1991 in xe2x80x9cReproductive Endocrinologyxe2x80x9d, Eds. Yen, Jaffe and Benton, Pub. W B Saunders, Philadelphia; Baird, 1993 British Medical Bulletin 49, 73-87). Despite the undoubted success of this approach, no conceptual advances in contraceptive technology have arisen for 20 years, no means identified to improve implantation, no advances made in promoting placental growth and development and no new approaches found to treat benign gynaecological disease (menstrual dysfunction and fibroids).
A number of publications have been made in relation to the use of xe2x80x9cgene transferxe2x80x9d in mammals to alter the genotype of at least some cells in a certain tissue or tissues. In particular, it is known to attempt xe2x80x9cgene therapyxe2x80x9d of humans by the introduction into recipients of nucleic acid sequences, with the aim of overcoming a genetic deficiency in the recipient by the expression of polypeptides encoded by the introduced nucleic acid sequences. Gene therapy trials have been conducted, for example, in which DNA sequences (incorporated within viral vectors) were introduced into the airways of cystic fibrosis patients, so as to alter the phenotype of at least some of the epithelial cells lining the patients"" respiratory tract. Thus far, there have been no published attempts to introduce DNA into the mammalian endometrium, despite the availability of suitable techniques therefor.
In one aspect, the invention provides a method of altering one or more characteristics of at least some of the cells of the reproductive tract of a mammalian individual by the introduction into said cells of a nucleic acid.
In a second aspect the invention provides a composition comprising nucleic acid, for use in altering one or more characteristics of at least some of the cells of the reproductive tract of a mammalian individual.
In a third aspect the invention provides for use of a composition comprising nucleic acid for altering one or more characterstics of at least some of the cells of the reproductive tract of a mammalian individual.
In a fourth aspect the invention provides for use of a composition comprising nucleic acid in the preparation of a substance for altering one or more characteristics of at least some of the cells of the reproductive tract of a mammalian individual.
In a fifth aspect the invention provides a method of making a composition for use in altering one or more characteristics of at least some of the cells of the reproductive tract of a mammalian individual, comprising mixing a nucleic acid with a physiologically acceptable carrier substance.
The present invention can in no way be considered as an obvious extension of genetic therapy techniques already known to be at least partially successful when applied to the lungs of cystic fibrosis patients. Inherited genetic disorders are not thought to be responsible for any of the known diseases of the endometrium, so there would have been no incentive for those skilled in the art to apply gene therapy techniques to the endometrium. Further, the epithelium of the endometrium is of a different type (cuboidal, derived from coelomic epithelium) compared to lung epithelium (which is stratified) and therefore could not have been predicted to behave in an analagous manner. Moreover, at least in primates, there is cyclical shedding of the endometrial epithelium which would tend to cause the loss of any transfected cells. Finally, the inventors have found that there was no transfer of the introduced DNA into the organs of the mother, nor into the placenta of the embryo, either of which might have occurred and could have caused practical difficulties.
Typically the nucleic acid is introduced into a mammalian female (preferably a woman) and, in particular, into the endometrial cells thereof. Desirably the nucleic acid is introduced into the glandular epithelium of the endometrium. The nucleic acid may encode a polypeptide which is already naturally synthesised by the cells into which the nucleic acid is introduced, such that the level of expression of that polypeptide is increased via a gene dosage effect. Alternatively the method can be used to induce the cells to express a polypeptide not previously synthesised in those cells. The polypeptide could, for example, be an xe2x80x9cartificialxe2x80x9d recombinant polypeptide which does not exist in nature, such as a chimeric polypeptide comprising, wholly or in part, functional domains from two or more different proteins.
The nucleic acid is preferably DNA, but one could seek to introduce RNA (either sense or non-sense strands). An antisense molecule could be used to inhibit or otherwise interfere with the expression of a polypeptide in the cells into which the nucleic acid is introduced. The nucleic acid sequence introduced may be antisense RNA, or may be a DNA sequence directing the synthesis, intracellularly, of antisense RNA. Another way of achieving such inhibition is to introduce into the cells a sequence directing the synthesis of a ribozyme, which will then specifically cleave the mRNA needed to synthesise the polypeptide whose expression is sought to be inhibited.
The present inventors have found that the time of administration of the nucleic acid (relative to the stage of the reproductive cycle) greatly affects the efficiency of uptake of the nucleic acid. The inventors have found that, in general, in order to obtain the optimum degree of uptake of the administered nucleic acid it is necessary for the administration to be made in the period following ovulation, up to and including the day on which there is a peak of progesterone level in the blood. The progesterone level normally peaks at around a similar time to the point at which an embryo, if present in the uterus, could become implanted.
Thus, for example, the inventors have found that maximal uptake of administered DNA by mouse endometrium occurs at day 2-3 in the cycle (with day 1 taken as the day on which a vaginal plug is first detected). In humans, ovulation typically occurs at day 14 of the cycle, and implantation is generally reckoned to occur in the mid-luteal phase (although the exact time is poorly defined in humans).
The nucleic acid may be administered in a naked form, or may be bound or associated with other substances (e.g. liposomes). Conveniently the nucleic acid is introduced into the cells of the recipient mammal by simple transfection (with or without liposomes), which has been found by the present inventors to be surprisingly effective, without the need for the sequence to be introduced within a viral vector. Nevertheless, viral vectors may be desirable, especially those which may be targeted to certain cell types (e.g. as disclosed in WO 93/20221).
The nucleic acid will conveniently be introduced as part of a construct (e.g. a plasmid, cosmid or the like), which construct will advantageously comprise a promoter, operable in a mammal, to cause transcription of at least part of the introduced nucleic acid. The promoter may be constitutive or, more preferably, inducible so as to allow greater control of expression of the introduced sequence.
In one particular method performed in accordance with the invention, introduction of a nucleic acid molecule into the endometrial cells of an individual mammalian female allows for the up- or down-regulation of the fertility of the individual. The invention may particularly be used to provide a method of contraception for companion animals (e.g. cats and dogs) to prevent unwanted litters. In other embodiments the invention provides a method of improving the fertility of livestock species, such as pigs, cattle, sheep and the like.
Preferably the nucleic acid is introduced into the reproductive tract via the vagina, which avoids the need for invasive surgical techniques. However, if necessary, the nucleic acid could be introduced by means of surgical techniques directly into the reproductive tract (e.g. into the uterus). The invention offers the possibility of altering one or more characteristics by the introduction of one or more of a very large number of different nucleic acid sequences.
In one embodiment, the sequence introduced into the reproductive tract cells directs the expression (preferably at high levels) of an effective portion of a cytokine or growth factor (an effective portion is that part of the molecule which retains the biological activity particularly associated with the whole e.g. binding to a specific ligand etc.). Examples of such polypeptides which might be expressed by the introduced sequence include, but are not limited to, the following: interleukins, leukaemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), heparin-binding epidermal growth factor (HBEGF), insulin-binding growth factors I and II (IGF-I and IGF-II), amphiregulin, colony stimulating factor (CSF), and tumour necrosis factor (TNF).
In another embodiment the introduced sequence may direct the expression of an effective portion of an antagonist of a cytokine or growth factor, such as the IL-1 receptor antagonist. Advantageously, the antagonist may be a soluble receptor for the cytokine or growth factor. Suitable examples include soluble receptors for the following: transforming growth factor (TGF) xcex1, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), interleukin-6 (IL-6), and VEGF.
In another embodiment the introduced sequence may direct the expression of an effective portion of a polypeptide having an immunological effect. In particular, the polypeptide may possess immunogenic activity, thereby serving to stimulate a local immune response, thus the invention can be used to provide a novel method of immunisation. Advantageously the immunogenic polypeptide will be an antigen from a mucosal pathogen. By virtue of the common mucosal immune system, stimulation of antibody production in the reproductive tract may result in the production of corresponding antibodies at distal mucosal sites, such as the gastro-intestinal tract, the respiratory tract, lachrymal glands and the like. Preferably however the antigen will be one from a pathogen which invades and/or colonises the reproductive tract, typically a pathogen which causes a sexually transmitted disease. Examples include viruses such as HIV, papilloma viruses (e.g. HPV, of various types), chlamydia and bacteria (e.g. N. gonorrhoea). Alternatively, the polypeptide having an immunoloigical effect may be an immunoglobulin or effective portion thereof (such as an Fab, Fv, or scFv fragment, or a single chain antibody). The immunoglobulin or effective portion thereof may be directed against a pathogen (such as those mentioned above), or may be directed against some other antigen, such as a steroid or other hormone. Thus immunoglobulins or fragments thereof could be expressed locally to provide protection against disease or to regulate fertilty.
In another embodiment, the introduced sequence may direct the expression of a polypeptide, or an effective portion thereof, which has an effect on menstruation.
In another embodiment the introduced nucleic acid may direct the expression, on the surface of the reproductive tract cells, of an effective portion of a receptor molecule. The receptor could be a receptor for a cytokine, a steroid hormone, or a growth factor (such as the EGF receptor, the TGFxcex1 receptor, or the VEGF receptor). A number of receptors are known which are described as xe2x80x9corphanxe2x80x9d receptors, in that the ligand which binds to the receptor is unknown. Such orphan receptors are of considerable interest to the pharmaceutical industry, as they may provide targets for novel therapeutic or prophylactic compounds.
Accordingly, in another aspect the invention provides a method of characterising the biological properties of a polypeptide, comprising introducing the sequence encoding the polypeptide to be characterised into the cells of the reproductive tract of a mammal, and assessing the effects of the expressed polypeptide. Preferably the mammal is a laboratory animal, such as a mouse or rat. Conveniently, the polypeptide to be characterised will be an orphan receptor and typically at least part of the characterisation thereof will comprise identification of the ligand therefor. Generally the method will involve the analysis of histological sections taken from the laboratory mammal, and processing thereof by any one of various standard techniques (e.g. histochemical staining, in situ hybridisation, immunological staining etc.).
The present invention thus offers a novel alternative to steroidal regulation of endometrial function (and thus reproductive capacity or fertility) by direct gene transfer in vivo. To achieve this, genetic constructs would be designed to specifically modulate cytokine action. This can be achieved in a variety of ways. For example the cells that produce a secreted cytokine could be prevented from synthesizing the factor by blocking transcription and translation using promoter driven antisense constructs or ribozymes. Alternatively the action of the secreted factor can be blocked by receptor antagonists. Naturally occurring soluble receptors may scavenge and neutralize bioactive ligand thereby acting as competitive receptor antagonists. Alternatively there are natural receptor antagonists, for example IL1Ra (interleukin-1 receptor antagonist). Intraperitoneal administration of this protein blocks blastocyst implantation in the mouse (Simon et al., 1994 cited elsewhere).
There is considerable evidence to show that soluble growth factors secreted by the oviduct and uterine epithelium can control pre-implantation development of the mammalian embryo, by acting directly through receptors expressed on the embryo (Pampfer et al., 1990 In Vitro Cellular and Developmental Biology 26, 944-948). In turn, developing embryos produce growth factors which may act in an autocrine fashion, or on the endometrium to influence its receptivity. For example, in mice, LIF expression (from maternal tissues) is dramatically upregulated in glandular epithelium on day 4, just prior to implantation. LIF is able to act on pre-implantation blastocysts, which express the LIF receptor (LIF-R). This maternal expression of LIF is vital for implantation since in LIF knockout mice, embryos will not implant, although they will do so on transfer to pseudopregnant dams (Stewart et al., 1992 cited elsewhere).
The inventors have now extended this work to humans, and shown by RT-PCR that human embryos express the mRNA encoding the LIF-R, but do not themselves express LIF. LIF acts by binding to a low affinity receptor LIF-R. High affinity binding arises when the LIF/LIF-R complex interacts with the signal transducing accessory protein gp130. Human embryos also contain mRNA encoding this protein (Sharkey et al., 1995 Biology of Reproduction 53, 955-962). The inventors have also shown LIF secretion in human glandular epithelium is regulated by steroids, being maximal in the luteal phase (around the expected time of implantationxe2x80x94Charnock-Jones et al., 1994 cited elsewhere). Furthermore, administration of LIF to human pre-implantation embryos in vitro, has been reported to improve development. All this evidence supports the idea that LIF may be important in human implantation as it is in the mouse. Clearly cytokines may mediate important communication between the embryo in the uterine lumen, and the endometrium (in both directions) The present invention allows the use of gene transfer to disrupt or enhance this communication, leading to novel methods of contraception, or conversely improved implantation.
Most current studies of the paracrine and autocrine regulation of reproductive function are limited to a descriptive analysis by the lack of effective methods to modulate local cytokine/receptor levels. The evidence presented in this application indicates that transfection of uterine epithelium in vitro is feasible. This allows the endometrium to be manipulated experimentally and offers new therapeutic strategies. The work outlined below describes the use of a reporter gene to demonstrate the practicability of in vivo uterine gene transfer. In practice a gene (or other DNA construct), able to alter uterine function, would be used. Examples of these include receptor antagonists (e.g. IL-1Ra, soluble VEGF receptors etc.) natural or modified cytokines and growth factors, protease inhibitors or steroid receptors and a variety of ribozyme and antisense constructs. This work shows that genes can be transferred to the endometrium in vivo and this will find utility in many endometrial (and placental) conditions for example improving implantation in both animals and man, disrupting implantation (i.e. contraception), endometriosis and menorrhagia, hyperplasia and adenocarcinoma.
Using the protocols we have developed the results described below were obtained. They demonstrate that gene constructs can be transferred to the endometrium both in vivo (in mice) and in vitro and that these constructs are transcriptionally (and translationally) active.